Antibodies to human il-1beta

ABSTRACT

An IL-1β binding molecule, in particular an antibody to human IL-1β, especially a human antibody to human IL-1β is provided, wherein the CDRs of the heavy and light chains having amino acid sequences as defined, for use in the treatment of an IL-1 mediated disease or disorder, e.g. osteoarthritis, osteoporosis and other inflammatory arthritides.

[0001] This invention relates to antibodies to human interleukin I beta(IL-1β) and to the use of such antibodies for the treatment of IL-1mediated diseases and disorders.

[0002] Interleukin 1 (IL-1) is an activity produced by cells of theimmune system which acts as a mediator of the acute phase inflammatoryresponse. Inappropriate or excessive production of IL-1, in particularIL-1β, is associated with the pathology of various diseases anddisorders, such as septicemia, septic or endotoxic shock, allergies,asthma, bone loss, ischemia, stroke, rheumatoid arthrititis and otherinflammatory disorders. Antibodies to IL-1β have been proposed for usein the treatment of IL-1 mediated diseases and disorders; see forinstance, WO 95/01997 and the discussion in the introduction thereof.

[0003] We have now prepared improved antibodies to human IL-1β for usein the treatment of IL-1 mediated diseases and disorders.

[0004] Accordingly the invention provides an IL-1β binding moleculewhich comprises an antigen binding site comprising at least oneimmunoglobulin heavy chain variable domain (V_(H)) which comprises insequence hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having theamino acid sequence Val-Tyr-Gly-Met-Asn, said CDR2 having the amino acidsequenceile-ileTrp-Tyr-Asp-Gly-Asp-Asn-Gln-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly, andsaid CDR3 having the amino acid sequence Asp-Leu-Arg-Thr-Gly-Pro; anddirect equivalents thereof.

[0005] Accordingly the invention also provides an IL-1β binding moleculecomprising at least one immunoglobulin light chain variable domain(V_(L)) which comprises in sequence hypervariable regions CDR1′, CDR2′and CDR3′, said CDR1′ having the amino acid sequenceArg-Ala-Ser-Gln-Ser-Ile-Gly-Ser-Ser-Leu-His said CDR2′ having the aminoacid sequence Ala-Ser-Gin-Ser-Phe-Ser and said CDR3′ having the aminoacid sequence His-Gln-Ser-Ser-Ser-Leu-Pro and direct equivalent thereof.

[0006] In a first aspect the invention provides a single domain IL-1βbinding molecule comprising an isolated immunoglobulin heavy chaincomprising a heavy chain variable domain (V_(H)) as defined above.

[0007] In a second aspect the invention also provides an IL-1β bindingmolecule comprising both heavy (V_(H)) and light chain (V_(L)) variabledomains in which said IL-1β binding molecule comprises at least oneantigen binding site comprising:

[0008] a) an immunoglobulin heavy chain variable domain (V_(H)) whichcomprises in sequence hypervariable regions CDR1, CDR2 and CDR3, saidCDR1 having the amino acid sequence Val-Tyr-Gly-Met-Asn, said CDR2having the amino acid sequenceBle-Ile-Trp-Tyr-Asp-Gly-Asp-Asn-Gln-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly, andsaid CDR3 having the amino acid sequence Asp-Leu-Arg-Thr-Gly-Pro, and

[0009] b) an immunoglobulin light chain variable domain (V_(L)) whichcomprises in sequence hypervariable regions CDR1′, CDR2′ and CDR3′, saidCDR1′ having the amino acid sequenceArg-Ala-Ser-Gln-Ser-Ile-Gly-Ser-Ser-Leu-His, said CDR2′ having the aminoacid sequence Ala-Ser-Gln-Ser-Phe-Ser, and said CDR3′ having the aminoacid sequence His-Gln-Ser-Ser-Ser-Leu-Pro;

[0010] and direct equivalents thereof.

[0011] Unless otherwise indicated, any polypeptide chain is hereindescribed as having an amino acid sequence starting at the N-terminalextremity and ending at the C-terminal extremity. When the antigenbinding site comprises both the V_(H) and V_(L) domains, these may belocated on the same polypeptide molecule or, preferably, each domain maybe on a different chain, the V_(H) domain being part of animmunoglobulin heavy chain or fragment thereof and the V_(L) being partof an immunoglobulin light chain or fragment thereof.

[0012] By “IL-1β binding molecule” is meant any molecule capable ofbinding to the IL-1β antigen either alone or associated with othermolecules. The binding reaction may be shown by standard methods(qualitative assays) including, for example, a bioassay for determiningthe inhibition of IL-1β binding to its receptor or any kind of bindingassays, with reference to a negative control test in which an antibodyof unrelated specificity but of the same isotype, e.g. an anti-CD25antibody, is used. Advantageously, the binding of the IL-1β bindingmolecules of the invention to IL-1β may be shown in a competitivebinding assay.

[0013] Examples of antigen binding molecules include antibodies asproduced by B-cells or hybridomas and chimeric, CDR-grafted or humanantibodies or any fragment thereof, e.g. F(ab′)₂ and Fab fragments, aswell as single chain or single domain antibodies.

[0014] A single chain antibody consists of the variable domains of theheavy and light chains of an antibody covalently bound by a peptidelinker usually consisting of from 10 to 30 amino acids, preferably from15 to 25 amino acids. Therefore, such a structure does not include theconstant part of the heavy and light chains and it is believed that thesmall peptide spacer should be less antigenic than a whole constantpart. By “chimeric antibody” is meant an antibody in which the constantregions of heavy or light chains or both are of human origin while thevariable domains of both heavy and light chains are of non-human (e.g.murine) origin or of human origin but derived from a different humanantibody. By “CDR-grafted antibody” is meant an antibody in which thehypervariable regions (CDRs) are derived from a donor antibody, such asa non-human (e.g. murine) antibody or a different human antibody, whileall or substantially all the other parts of the immunoglobulin e.g. theconstant regions and the highly conserved parts of the variable domains,i.e. the framework regions, are derived from an acceptor antibody, e.g.an antibody of human origin. A CDR-grafted antibody may however containa few amino acids of the donor sequence in the framework regions, forinstance in the parts of the framework regions adjacent to thehypervariable regions. By “human antibody” is meant an antibody in whichthe constant and variable regions of both the heavy and light chains areall of human origin, or substantially identical to sequences of humanorigin, not necessarily from the same antibody and includes antibodiesproduced by mice in which the murine immunoglobulin variable andconstant part genes have been replaced by their human counterparts, e.g.as described in general terms in EP 0546073 B1, U.S. Pat. No. 5,545,806,U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126, U.S. Pat. No.5,633,425, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,770,429, EP 0 438474B1 and EP 0 463151 B1.

[0015] Particularly preferred IL-1β binding molecules of the inventionare human antibodies especially the ACZ 885 antibody as hereinafterdescribed in the Examples.

[0016] Thus in preferred chimeric antibodies the variable domains ofboth heavy and light chains are of human origin, for instance those ofthe ACZ 885 antibody which are shown in Seq. Id. No. 1 and Seq. Id. No.2. The constant region domains preferably also comprise suitable humanconstant region domains, for instance as described in “Sequences ofProteins of Immunological Interest”, Kabat E. A. et al, US Department ofHealth and Human Services, Public Health Service, National Institute ofHealth

[0017] Hypervariable regions may be associated with any kind offramework regions, though preferably are of human origin. Suitableframework regions are described in Kabat E. A. et al, ibid. Thepreferred heavy chain framework is a human heavy chain framework, forinstance that of the ACZ 885 antibody which is shown in Seq. Id. No. 1.It consists in sequence of FR1, FR2, FR3 and FR4 regions. In a similarmanner, Seq. Id. No. 2 shows the preferred ACZ 885 light chain frameworkwhich consists, in sequence, of FR1′, FR2′, FR3′ and FR4′ regions.

[0018] Accordingly, the invention also provides an IL-1β bindingmolecule which comprises at least one antigen binding site comprisingeither a first domain having an amino acid sequence substantiallyidentical to that shown in Seq. Id. No. 1 starting with the amino acidat position 1 and ending with the amino acid at position 118 or a firstdomain as described above and a second domain having an amino acidsequence substantially identical to that shown in Seq. Id. No. 2,starting with the amino acid at position 1 and ending with the aminoacid at position 107.

[0019] Monoclonal antibodies raised against a protein naturally found inall humans are typically developed in a non-human system e.g. in mice,and as such are typically non-human proteins. As a direct consequence ofthis, a xenogenic antibody as produced by a hybridoma, when administeredto humans, elicits an undesirable immune response which is predominantlymediated by the constant part of the xenogenic immunoglobulin. Thisclearly limits the use of such antibodies as they cannot be administeredover a prolonged period of time. Therefore it is particularly preferredto use single chain, single domain, chimeric, CDR-grafted, or especiallyhuman antibodies which are not likely to elicit a substantial allogenicresponse when administered to humans.

[0020] In view of the foregoing, a more preferred IL-1β binding moleculeof the invention is selected from a human anti IL-1β antibody whichcomprises at least

[0021] a) an immunoglobulin heavy chain or fragment thereof whichcomprises (i) a variable domain comprising in sequence the hypervariableregions CDR1, CDR2 and CDR3 and (ii) the constant part or fragmentthereof of a human heavy chain; said CDR1 having the amino acid sequenceVal-Tyr-Gly-Met-Asn, said CDR2 having the amino acid sequenceIle-IleTrp-Tyr-Asp-Gly-Asp-Asn-Gln-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly, andsaid CDR3 having the amino acid sequence Asp-Leu-Arg-Thr-Gly-Pro and

[0022] b) an immunoglobulin light chain or fragment thereof whichcomprises (i) a variable domain comprising in sequence the hypervariableregions and optionally also the CDR1′, CDR2′, and CDR3′ hypervariableregions and (ii) the constant part or fragment thereof of a human lightchain, said CDR1′ having the amino acid sequenceArg-Ala-Ser-Gln-Ser-Ile-Gly-Ser-Ser-Leu-His, said CDR2′ having the aminoacid sequence Ala-Ser-Gln-Ser-Phe-Ser, and said CDR3′ having the aminoacid sequence His-Gln-Ser-Ser-Ser-Leu-Pro;

[0023] and direct equivalents thereof.

[0024] Alternatively, an IL-1β binding molecule of the invention may beselected from a single chain binding molecule which comprises an antigenbinding site comprising

[0025] a) a first domain comprising in sequence the hypervariableregions CDR1, CDR2 and CDR3, said hypervariable regions having the aminoacid sequences as shown in Seq. Id. No. 1,

[0026] b) A second domain comprising the hypervariable regions CDR1′,CDR2′ and CDR3′ said hypervariable regions having the amino acidsequences as shown in Seq. Id. No. 2 and

[0027] c) a peptide linker which is bound either to the N-terminalextremity of the first domain and to the C-terminal extremity of thesecond domain or to the C-terminal extremity of the first domain and tothe N-terminal extremity of second domain;

[0028] and direct equivalents thereof.

[0029] As it is well known, minor changes in an amino acid sequence suchas deletion, addition or substitution of one, a few or even severalamino acids may lead to an allelic form of the original protein whichhas substantially identical properties.

[0030] Thus, by the term “direct equivalents thereof” is meant eitherany single domain IL-1β binding molecule (molecule X).

[0031] (i) in which the hypervariable regions CDR1, CDR2 and CDR3 takenas a whole are at least 80% homologous, preferably at least 90%homologous, more preferably at least 95% homologous to the hypervariableregions as shown in Seq. Id. No. 1 and,

[0032] (ii) which is capable of inhibiting the binding of IL-1β to itsreceptor substantially to the same extent as a reference molecule havingframework regions identical to those of molecule X but havinghypervariable regions CDR1, CDR2 and CDR3 identical to those shown inSeq. Id. No. 1

[0033] or any IL-1β binding molecule having at least two domains perbinding site (molecule X′)

[0034] (i) in which the hypervariable regions CDR1, CDR2, CDR3, CDR1′,CDR2′ and CDR3′ taken as a whole are at least 80% homologous, preferablyat least 90% homologous, more preferably at least 95% homologous, to thehypervariable regions as shown in Seq. Id. No. 1 and 2 and

[0035] (ii) which is capable of inhibiting the binding of IL-1β to itsreceptor substantially to the same extent as a reference molecule havingframework regions and constant parts identical to molecule X′, buthaving hypervariable regions CDR1, CDR2, CDR3, CDR1′, CDR2′ and CDR3′,identical to those shown in Seq. Id. No. 1 and 2.

[0036] In the present description amino acid sequences are at least 80%homologous to one another if they have at least 80% identical amino acidresidues in a like position when the sequence are aligned optimally,gaps or insertions in the amino acid sequences being counted asnon-identical residues.

[0037] The inhibition of the binding of IL-1β to its receptor may beconveniently tested in various assays including such assays aredescribed hereinafter in the text. By the term “to the same extent” ismeant that the reference and the equivalent molecules exhibit, on astatistical basis, essentially identical IL-1β binding inhibition curvesin one of the assays referred to above. For example, in IL-1β bindingmolecules of the invention typically have IC₅₀s for the inhibition ofthe binding of IL-1β to its receptor which are within +/−x5 of that of,preferably substantially the same as, the IC₅₀ of the correspondingreference molecule when assayed as described above.

[0038] For example, the assay used may be an assay of competitiveinhibition of binding of IL-1β by soluble IL-1 receptors and the IL-1βbinding molecules of the invention.

[0039] Most preferably, the human IL-1β antibody comprises at least

[0040] a) one heavy chain which comprises a variable domain having anamino acid sequence substantially identical to that shown in Seq. Id.No. 1 starting with the amino acid at position 1 and ending with theamino acid at position 118 and the constant part of a human heavy chain;and

[0041] b) one light chain which comprises a variable domain having anamino acid sequence substantially identical to that shown in Seq. Id.No. 2 starting with the amino acid at position 1 and ending with theamino acid at position 107 and the constant part of a human light chain.

[0042] The constant part of a human heavy chain may be of the γ1, γ2,γ3, γ4, μ, β2, or δ or ε type, preferably of the γ type, more preferablyof the y, type, whereas the constant part of a human light chain may beof the κ or λ type (which includes the λ₁, λ₂ and λ₃ subtypes) but ispreferably of the κ type. The amino acid sequences of all these constantparts are given in Kabat et al ibid.

[0043] An IL-1β binding molecule of the invention may be produced byrecombinant DNA techniques. In view of this, one or more DNA moleculesencoding the binding molecule must be constructed, placed underappropriate control sequences and transferred into a suitable hostorganism for expression.

[0044] In a very general manner, there are accordingly provided

[0045] (i) DNA molecules encoding a single domain ILIAD bindingmolecule, of the invention, a single chain IL-1β binding molecule of theinvention, a heavy or light chain or fragments thereof of a IL-1βbinding molecule of the invention and

[0046] (ii) the use of the DNA molecules of the invention for theproduction of a IL-1β binding molecule of the invention by recombinantmeans.

[0047] The present state of the art is such that the skilled worker inthe art is able to synthesize the DNA molecules of the invention giventhe information provided herein i.e. the amino acid sequences of thehypervariable regions and the DNA sequences coding for them. A methodfor constructing a variable domain gene is for example described in EPA239 400 and may be briefly summarized as follows: A gene encoding avariable domain of a MAb of whatever specificity is cloned. The DNAsegments encoding the framework and hypervariable regions are determinedand the DNA segments encoding the hypervariable regions are removed sothat the DNA segments encoding the framework regions are fused togetherwith suitable restriction sites at the junctions. The restriction sitesmay be generated at the appropriate positions by mutagenesis of the DNAmolecule by standard procedures. Double stranded synthetic CDR cassettesare prepared by DNA synthesis according to the sequences given in Seq.Id. No. 1 or 2. These cassettes are provided with sticky ends so thatthey can be ligated at the junctions of the framework

[0048] Furthermore, it is not necessary to have access to the mRNA froma producing hybridoma cell line in order to obtain a DNA constructcoding for the IL-1β binding molecules of the invention. Thus PCTapplication WO 90/07861 gives full instructions for the production of anantibody by recombinant DNA techniques given only written information asto the nucleotide sequence of the gene. The method comprises thesynthesis of a number of oligonucleotides, their amplification by thePCR method, and their splicing to give the desired DNA sequence.

[0049] Expression vectors comprising a suitable promoter or genesencoding heavy and light chain constant parts are publicly available.Thus, once a DNA molecule of the invention is prepared it may beconveniently transferred in an appropriate expression vector. DNAmolecules encoding single chain antibodies may also be prepared bystandard methods, for example, as described in WO 88/1649.

[0050] In view of the foregoing no hybridoma or cell line deposit isnecessary to comply with the criteria of sufficiency of description.

[0051] In a particular embodiment the invention includes first andsecond DNA constructs for the production of an IL-1β binding molecule asdescribed below:

[0052] The first DNA construct encodes a heavy chain or fragment thereofand comprises

[0053] a) a first part which encodes a variable domain comprisingalternatively framework and hypervariable regions, said hypervariableregions being in sequence CDR1, CDR2 and CDR3 the amino acid sequencesof which are shown in Seq. Id. No. 1; this first part starting with acodon encoding the first amino acid of the variable domain and endingwith a codon encoding the last amino acid of the variable domain, and

[0054] b) a second part encoding a heavy chain constant part or fragmentthereof which starts with a codon encoding the first amino acid of theconstant part of the heavy chain and ends with a codon encoding the lastamino acid of the constant part or fragment thereof, followed by a stopcodon.

[0055] Preferably, this first part encodes a variable domain having anamino acid sequence substantially identical to the amino acid sequenceas shown in Seq. Id. No. 1 starting with the amino acid at position 1and ending with the amino acid at position 118. More preferably thefirst part has the nucleotide sequence as shown in Seq. Id. No. 1starting with the nucleotide at position 1 and ending with thenucleotide at position 354. Also preferably, the second part encodes theconstant part of a human heavy chain, more preferably the constant partof the human γ1 chain. This second part may be a DNA fragment of genomicorigin (comprising introns) or a cDNA fragment (without introns).

[0056] The second DNA construct encodes a light chain or fragmentthereof and comprises

[0057] a) a first part which encodes a variable domain comprisingalternatively framework and hypervariable regions; said hypervariableregions being CDR3′ and optionally CDR1′ and CDR2′, the amino acidsequences of which are shown in Seq. Id. No. 2; this first part startingwith a codon encoding the first amino acid of the variable domain andending with a codon encoding the last amino acid of the variable domain,and

[0058] b) a second part encoding a light chain constant part or fragmentthereof which starts with a codon encoding the first amino acid of theconstant part of the light chain and ends with a codon encoding the lastamino acid of the constant part or fragment thereof followed by a stopcodon.

[0059] Preferably, this first part encodes a variable domain having anamino acid sequence substantially identical to the amino acid sequenceas shown in Seq. Id. No. 2 starting with the amino acid at position 1and ending with the amino acid at position 107. More preferably, thefirst part has the nucleotide sequence as shown in Seq. Id. No. 2starting with the nucleotide at position 1 and ending with thenucleotide at position 321. Also preferably the second part encodes theconstant part of a human light chain, more preferably the constant partof the human κ chain.

[0060] The invention also includes IL-1β binding molecules in which oneor more of the residues of CDR1, CDR2, CDR3, CDR1, CDR2′ or CDR3′ or theframeworks, typically only a few (e.g. 1-4), are changed from theresidues shown in Seq Id No. 1 and Seq. Id. No. 2; for instance bymutation e.g. site directed mutagenesis of the corresponding DNAsequences. The invention includes the DNA sequences coding for suchchanged IL-1β binding molecules. In particular the invention includesIL-1β binding molecules in which one or more residues of CDR1′ or CDR2′have been changed from the residues shown in Seq. Id. No. 2.

[0061] In the first and second DNA constructs, the first and secondparts may be separated by an intron, and, an enhancer may beconveniently located in the intron between the first and second parts.The presence of such an enhancer which is transcribed but nottranslated, may assist in efficient transcription. In particularembodiments the first and second DNA constructs comprise the enhancer ofa heavy chain gene advantageously of human origin.

[0062] Each of the DNA constructs are placed under the control ofsuitable control sequences, in particular under the control of asuitable promoter. Any kind of promoter may be used, provided that it isadapted to the host organism in which the DNA constructs will betransferred for expression. However, if expression is to take place in amammalian cell, it is particularly preferred to use the promoter of animmunoglobulin gene.

[0063] The desired antibody may be produced in a cell culture or in atransgenic animal. A suitable transgenic animal may be obtainedaccording to standard methods which include micro injecting into eggsthe first and second DNA constructs placed under suitable controlsequences transferring the so prepared eggs into appropriatepseudo-pregnant females and selecting a descendant expressing thedesired antibody.

[0064] When the antibody chains are produced in a cell culture, the DNAconstructs must first be inserted into either a single expression vectoror into two separate but compatible expression vectors, the latterpossibility being preferred.

[0065] Accordingly, the invention also provides an expression vectorable to replicate in a prokaryotic or eukaryotic cell line whichcomprises at least one of the DNA constructs above described.

[0066] Each expression vector containing a DNA construct is thentransferred into a suitable host organism. When the DNA constructs areseparately inserted on two expression vectors, they may be transferredseparately, i.e. one type of vector per cell, or co-transferred, thislatter possibility being preferred. A suitable host organism may be abacterium, a yeast or a mammalian cell line, this latter beingpreferred. More preferably, the mammalian cell line is of lymphoidorigin, e.g. a myeloma, hybridoma or a normal immortalised B-cell, whichconveniently does not express any endogenous antibody heavy or lightchain.

[0067] For expression in mammalian cells it is preferred that the IL-1βbinding molecule coding sequence is integrated into the host cell DNAwithin a locus which permits or favours high level expression of theIL-1β binding molecule. Cells in which the IL-1β binding molecule codingsequence is integrated into such favourable loci may be identified andselected on the basis of the levels of the IL-1β binding molecule whichthey express. Any suitable selectable marker may be used for preparationof host cells containing the IL-1β binding molecule coding sequence; forinstance, a dhfr gene/methotrexate or equivalent selection system may beused. Alternative systems for expression of the IL-1β binding moleculesof the invention include GS-based amplification/selection systems, suchas those described in EP 0256055 B, EP 0323997 B and European patentapplication 89303964.4.

[0068] In a further aspect of the invention there is provided a processfor the product of an IL-1β binding molecule which comprises (i)culturing an organism which is transformed with an expression vector asdefined above and (ii) recovering the IL-1β binding molecule from theculture.

[0069] In accordance with the present invention it has been found thatthe ACZ 885 antibody appears to have binding specificity for theantigenic epitope of human IL-1β which includes the loop comprising theGlu 64 residue of mature human IL-1β. (Residue Glu 64 of mature humanIL-1β correspond to residue 180 of the human IL-1β precursor.) Thisepitope appears to be outside the recognition site of the IL-1 receptorand it is therefore most surprising that antibodies to this eptitope,e.g. the ACZ 885 antibody, are capable of inhibiting the binding ofIL-1β to its receptor. Antibodies, in particular chimeric andCDR-grafted antibodies and especially human antibodies, which havebinding specificity for the antigenic epitope of mature human IL-1βwhich includes the loop comprising residue Glu 64 and which are capableof inhibiting the binding of IL-1β to its receptor; and use of suchantibodies for the treatment of IL-1 mediated diseases and disorders,are novel and are included within the scope of the present invention.

[0070] Thus in a further aspect the invention includes an antibody toIL-1β which has antigen binding specificity for an antigenic epitope ofhuman IL-1β which includes the loop comprising residue Glu 64 of maturehuman IL-1β and which is capable of inhibiting the binding of IL-1β toits receptor.

[0071] In yet further aspects the invention includes:

[0072] i) use of an antibody to IL-1β, which has antigen bindingspecificity for an antigenic epitope of mature human IL-1β whichincludes the loop comprising Glu 64 and which is capable of inhibitingthe binding of IL-1β to its receptor, for the treatment of an IL-1mediated disease or disorder;

[0073] ii) a method for the treatment of an IL-1 mediated disease ordisorders in a patient which comprises administering to the patient aneffective amount of an antibody to IL-1β, which has antigen bindingspecificity for an antigenic epitope of mature human IL-1β whichincludes the loop comprising Glu 64 and which is capable of inhibitingthe binding of IL-1β to its receptor;

[0074] iii) a pharmaceutical composition comprising an antibody toIL-1β, which has antigen binding specificity for an antigenic epitope ofmature human IL-1β which includes the loop comprising Glu 64 and whichis capable of inhibiting the binding of IL-1β to its receptor, incombination with a pharmaceutically acceptable excipient, diluent orcarrier; and

[0075] iv) use of an antibody to IL-1β, which has antigen bindingspecificity for an antigenic epitope of mature human IL-1β whichincludes the loop comprising Glu 64 and which is capable of inhibitingthe binding of IL-1β to its receptor, for the preparation of amedicament for the treatment of an IL-1 mediated disease or disorder.

[0076] For the purposes of the present description an antibody is“capable of inhibiting the binding of IL-1β” if the antibody is capableof inhibiting the binding of IL-1β to its receptor substantially to thesame extent as the ACZ 885 antibody, wherein “to the same extent” hasmeaning as defined above.

[0077] The ACZ 885 antibody has binding affinity for IL-1β which ishigher than affinities previously reported for anti-IL-1β antibodiese.g. anti human IL-1β antibodies. Thus ACZ 885 has a dissociationequilibrium constant K_(D) for binding to IL-1β of less than about 50pM, e.g. about 35 pM. This high binding affinity makes the ACZ antibodyparticularly suitable for therapeutic applications.

[0078] Thus in a yet further aspect the invention provides an antibodyto IL-1β which has a K_(D) for binding to IL-1β of about 50 pM or less.This aspect of the invention also includes uses methods and compositionsfor such high affinity antibodies, as described above for antibodies toIL-1β have binding specificity for an antigenic determinant of maturehuman IL-1β which includes the loop comprising Glu 64.

[0079] In the present description the phrase “Il-1 mediated disease”encompasses all diseases and medical conditions in which IL-1 plays arole, whether directly or indirectly, in the disease or medicalcondition, including the causation, development, progress, persistenceor pathology of the disease or condition.

[0080] In the present description the terms “treatment” or “treat” referto both prophylactic or preventative treatment as well as curative ordisease modifying treatment, including treatment of patient at risk ofcontracting the disease or suspected to have contracted the disease aswell as patients who are ill or have been diagnosed as suffering from adisease or medical condition, and includes suppression of clinicalrelapse.

[0081] IL-1β binding molecules as defined above, in particular IL-1βbinding molecules according to the first and second aspects of theinvention antibodies which have binding specificity for the antigenicepitope of mature human IL-1β which includes the loop comprising Glu 64,in particular antibodies which are capable of inhibiting the binding ofIL-1β to its receptor; and antibodies to IL-1β which have a K_(D) forbinding to IL-1β of about 50 pM or less are herein referred to asAntibodies of the Invention.

[0082] Preferably the Antibodies of the Invention are IL-1β bindingmolecules according to the first and second aspects of the invention.Advantageously the Antibodies of the Invention are human antibodies,most preferably the ACZ 885 antibody or direct equivalent thereof.

[0083] The Antibodies of the Invention block the effects of IL-1β on itstarget cells and thus are indicated for use in the treatment of IL-1mediated diseases and disorders. These and other pharmacologicalactivities of the Antibodies of the Invention may be demonstrated instandard test methods for example as described below:

[0084] Neutralization of IL-1β Dependent Production of PGE₂ andInterleukin-6 by Primary Human Fibroblasts

[0085] The production of PGE₂ and IL 6 in primary human dermalfibroblasts is dependent on IL-1β. TNF-α alone cannot efficiently inducethese inflammatory mediators, but synergizes with IL-1. Primary dermalfibroblasts are used as a surrogate model for IL-1β induced cellularactivation.

[0086] Primary human fibroblasts are stimulated with recombinant IL-1βor conditioned medium obtained from LPS-stimulated human PBMCs in thepresence of various concentrations of Antibody of the Invention orIL-1RA ranging from 6 to 18,000 pM. The chimeric anti-CD25 antibodySimulect® (basilixiab) is used as a matched isotype control. Supernatantis taken after 16 h stimulation and assayed for IL-6 by ELISA.Antibodies of the Invention typically have IC₅₀s for inhibition of IL-6production of about 1 nM or less (e.g. from about 0.1 to about 1 nM)when tested as above.

[0087] As indicated in the above assay Antibodies of the Inventionpotently block the effects of IL-1β. Accordingly, the Antibodies of theInvention have pharmaceutical utility as follows:

[0088] Antibodies of the Invention are useful for the prophylaxis andtreatment of IL-1 mediated diseases or medical conditions, e.g.inflammatory conditions, allergies and allergic conditions,hypersensitivity reactions, autoimmune diseases, severe infections, andorgan or tissue transplant rejection.

[0089] For example, Antibodies of the Invention may be use for thetreatment of recipients of heart, lung, combined heart-lung, liver,kidney, pancreatic, skin or corneal transplants, including allograftrejection or xenograft rejection, and for the prevention ofgraft-versus-host disease, such as following bone marrow transplant, andorgan transplant associated arteriosclerosis.

[0090] Antibodies of the Invention are particularly useful for thetreatment, prevention, or amelioration of autoimmune disease and ofinflammatory conditions, in particular inflammatory conditions with anaetiology including an autoimmune component such as arthritis (forexample rheumatoid arthritis, arthritis chronica progrediente andarthritis deformans) and rheumatic diseases, including inflammatoryconditions and rheumatic diseases involving bone loss, inflammatorypain, hypersensitivity (including both airways hypersensitivity anddermal hypersensitivity) and allergies. Specific auto-immune diseasesfor which Antibodies of the Invention may be employed include autoimmunehaematological disorders (including e.g. hemolytic anaemia, aplasticanaemia, pure red cell anaemia and idiopathic thrombocytopenia),systemic lupus erythematosus, polychondritis, sclerodoma, Wegenergranulomatosis, dermatomyositis, chronic active hepatitis, myastheniagravis, psoriasis, Steven-Johnson syndrome, idiopathic sprue, autoimmuneinflammatory bowel disease (including e.g. ulcerative colitis, Crohn'sdisease and Irritable Bowel Syndrome), endocrine ophthalmopathy, Gravesdisease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis,juvenile diabetes (diabetes mellitus type I), uveitis (anterior andposterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis,interstitial lung fibrosis, psoriatic arthritis and glomerulonephritis(with and without nephrotic syndrome, e.g. including idiopathicnephrotic syndrome or minimal change nephropathy).

[0091] Antibodies of the Invention are also useful for the treatment,prevention, or amelioration of asthma, bronchitis, pneumoconiosis,pulmonary emphysema, and other obstructive or inflammatory diseases ofthe airways

[0092] Antibodies of the Invention are useful for treating undesirableacute and hyperacute inflammatory reactions which are mediated by IL-1or involve IL-1 production, especially IL-1β, or the promotion of TNFrelease by IL-1, e.g. acute infections, for example septic shock (e.g.,endotoxic shock and adult respiratory distress syndrome), meningitis,pneumonia; and severe burns; and for the treatment of cachexia orwasting syndrome associated with morbid TNF release, consequent toinfection, cancer, or organ dysfunction, especially AIDS-relatedcachexia, e.g., associated with or consequential to HIV infection.

[0093] Antibodies of the Invention are particularly useful for treatingdiseases of bone metabolism including osteoarthritis, osteoporosis andother inflammatory arthritides, and bone loss in general, includingage-related bone loss, and in particular periodontal disease.

[0094] For these indications, the appropriate dosage will, of course,vary depending upon, for example, the particular Antibody of theInvention to be employed, the host, the mode of administration and thenature and severity of the condition being treated. However, inprophylactic use, satisfactory results are generally indicated to beobtained at dosages from about 0.05 mg to about 10 mg per kilogram bodyweight more usually from about 0.1 mg to about 5 mg per kilogram bodyweight. The frequency of dosing for prophylactic uses will normally bein the range from about once per week up to about once every 3 months,more usually in the range from about once every 2 weeks up to about onceevery 10 weeks, e.g. once every 4 to 8 weeks. Antibody of the Inventionis conveniently administered parenterally, intravenously, e.g. into theantecubital or other peripheral vein, intramuscularly, orsubcutaneously. A prophylactic treatment typically comprisesadministering the Antibody of the Invention once per month to once every2 to 3 months, or less frequently. Pharmaceutical compositions of theinvention may be manufactured in conventional manner. A compositionaccording to the invention is preferably provided in lyophilized form.For immediate administration it is dissolved in a suitable aqueouscarrier, for example sterile water for injection or sterile bufferedphysiological saline. If it is considered desirable to make up asolution of larger volume for administration by infusion rather as abolus injection, it is advantageous to incorporate human serum albuminor the patient's own heparinised blood into the saline at the time offormulation. The presence of an excess of such physiologically inertprotein prevents loss of antibody by adsorption onto the walls of thecontainer and tubing used with the infusion solution. If albumin isused, a suitable concentration is from 0.5 to 4.5% by weight of thesaline solution.

[0095] The invention is further described by way of illustration in thefollowing Examples which refer to the accompanying Figure which showsdose response curves for the inhibition of IL-1β binding by soluble IL-1receptors I and II.

EXAMPLES

[0096] Transgenic mice engineered to express the human IgG/κ repertoireinstead of the murine immunoglobulin repertoire (Fishwild et al., 1996,Nat Biotechnol., 14, 845-851) are used to generate antibodies to humanIL-1β. B cells from these mice are immortalized by standard hybridomatechnology and murine hybridoma cells are obtained which secrete thehuman IgG1/κ antibody ACZ 885

Example 1 Generation of the Hybridoma and Purification of the Antibody

[0097] Genetically engineered mouse 18077 (Medarex Inc. Annadale, N.J.)is immunized with recombinant human IL-1β coupled to KLH (50 μg) s.c. inseveral sites in adjuvant. The mouse is boosted five additional timeswith the last injection three days before the fusion. On the day of thefusion mouse 18077 is killed by CO₂ inhalation and spleen cells(4.1×10⁷) are fused by a routine method using PEG 4000 with an equalnumber of PAI-O cells, a mouse myeloma cell line. Fused cells are platedout in 624 wells (1 ml/well) containing a feeder layer of mouseperitoneal cells (Balb C mice), in HAT supplemented RPMI 1640, 10% heatinactivated fetal calf serum 5×10⁻⁵ M β-mercaptoethanol. Supernatantsare collected and tested in ELISA and screened for IL-1β reactivemonoclonal antibodies. Five monoclonal antibodies of the IgG/κ subclassare identified. Cloning is done using 4×96 well microtiter plates,plating 0.5 cells per well. After two weeks wells are inspected with aninverted microscope. Supernatant is collected from wells positive forgrowth and production of anti-IL-1β monoclonal antibodies is evaluatedby ELISA. 1-2L of conditioned supernatant from four subclones of theoriginally identified hybridoma # 657 are prepared and antibodies arepurified by affinity chromatography on a protein A column.

[0098] Purity and Partial Amino Acid Sequences of Heavy and Light ChainAmino Acid Sequencing

[0099] Light and heavy chains of the purified antibody ACZ 885 areseparated by SDS-PAGE and the amino-terminal amino acids determined byEdman degradation. The purity of the antibody used in these studies is≧90% by sequencing. cDNA sequences coding for the heavy and light chainvariable domains are obtained by PCR amplification of cDNA obtained frommRNA from the cloned hybridoma cells and fully sequenced. Theamino-terminal sequences of heavy and light chain variable domains andthe corresponding DNA sequences are given in Seq. Id no. 1 and Seq IdNo. 2 below, in which the CDRs are shown in bold type. ACZ885 Heavychain variable region Seq. Id. No. 1ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGTCAG −19M  E  F  G  L  S  W  V  F  L  V  A  L  L  R  G  V  Q  C  Q −1GTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCV  Q  L  V  E  S  G  G  G  V  V  Q  P  G  R  S  L  R  L  S −21TGTGCAGCGTCTGGATTCACCTTCAGTGTTTATGGCATGAACTGGGTCCGCCAGGCTCCAC  A  A  S  G  F  T  F  S  V  Y  G  M  N  W  V  R  Q  A  P −41GGCAAGGGGCTGGAGTGGGTGGCAATTATTTGGTATGATGGAGATAATCAATACTATGCAG  K  G  L  E  W  V  A  I  I  W  Y  D  G  D  N  Q  Y  Y  A −61GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGD  S  V  K  G  R  F  T  I  S  R  D  N  S  K  N  T  L  Y  L −81CAAATGAACGGCCTGAGAGCCGAGGACACGGCTGTGTATTATTGTGCGAGAGATCTTAGGQ  M  N  G  L  R  A  E  D  T  A  V  Y  Y  C  A  R  D  L  R −101ACTGGGCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCT  G  P  F  D  Y  W  G  Q  G  T  L  V  T  V  S  S  −118 ACZ885 Lightchain variable region Seq. Id. No. 2ATGTTGCCATCACAACTCATTGGGTTTCTGCTGCTCTGGGTTCCAGCCTCCAGGGGTGAA −19M  L  P  S  Q  L  I  G  F  L  L  L  W  V  P  A  S  R  G  E −1ATTGTGCTGACTCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAGAAAGTCACCATCI  V  L  T  Q  S  P  D  F  Q  S  V  T  P  K  E  K  V  T  I −21ACCTGCCGGGCCAGTCAGAGCATTGGTAGTAGCTTACACTGGTACCAGCAGAAACCAGATT  C  R  A  S  Q  S  I  G  S  S  L  H  W  Y  Q  Q  K  P  D −41CAGTCTCCAAAGCTCCTCATCAAGTATGCTTCCCAGTCCTTCTCAGGGGTCCCCTCGAGGQ  S  P  K  L  L  I  K  Y  A  S  Q  S  F  S  G  V  P  S  R −61TTCAGTGGCAGTGGATCTGGGACAGATTTCACCCTCACCATCAATAGCCTGGAAGCTGAAF  S  G  S  G  S  G  T  D  F  T  L  T  I  N  S  L  E  A  E −81GATGCTGCAGCGTATTACTGTCATCAGAGTAGTAGTTTACCATTCACTTTCGGCCCTGGGD  A  A  A  Y  Y  C  H  Q  S  S  S  L  P  F  T  F  G  P  G −101ACCAAAGTGGATATCAAA T  K  V  D  I  K  −107

[0100] Italics: Leader sequence (not in mature antibody)

[0101] Bold: CDR's

[0102] Construction of Expression Vectors for Heavy and Light Chain

[0103] A GS based amplification/selection system such as that describedin EP 0256055 B, EP 0323997 B or European patent application 89303964.4is used, in which the selectable marker used is a GS coding sequence.

Example 2 Biochemical and Biological Data

[0104] The monoclonal antibody ACZ 885 is found to neutralize theactivity of interleukin-1β in vitro. The monoclonal antibody is furthercharacterized for its binding to recombinant human IL-1β Biacoreanalysis. The mode of neutralization is assessed by competitive bindingstudies with soluble IL-1 receptors. The biological activity of theantibody ACZ 885 towards recombinant and naturally produced IL-1β isdetermined in primary human cell (Example 3), responsive to stimulationby IL-1I.

[0105] Determination of Dissociation Equilibrium Constant

[0106] The association and dissociation rate constants for the bindingof recombinant human IL-1beta to ACZ885 are determined by BIAcoreanalysis. ACZ885 is immobilized, and binding of recombinant IL-1beta ina concentration range from 1 to 4 nM is measured by surface plasmonresonance. The chosen format represents a monovalent interaction andthus permits treating the binding event of IL-1 beta to ACZ885 accordingto a 1:1 stoichiometry. Data analysis is performed using theBIAevaluation software. k_(on) k_(off) K_(D) [10⁵/Ms] [10⁻⁵/s] [pM]Human IL-1β 11.0 +/− 0.23 3.3 +/− 0.27 30.5 +/− 2.6 n = 22

[0107] Conclusion: ACZ885 binds to recombinant human IL-1beta with veryhigh affinity.

[0108] Binding Competition Study with Soluble IL-1 type I and IIReceptors

[0109] Competition between ACZ885 and soluble human IL-1 type I and typeII receptors is measured by Biacore. ACZ885 is immobilized on the chipsurface and recombinant human IL-beta (1 nM) is injected for binding toACZ885 in absence or presence of increasing concentrations ofrecombinant human soluble receptor I or receptor 1 (0-12 nM; 4independent runs each.). The results obtained are given in theaccompanying Figure.

[0110] Binding of NVP-ACZ885 to human IL-1□ was determined in thepresence of recombinant human soluble IL-1 type I or type II receptors.Half-maximum values (IC₅₀) were determined graphically using the Origin6.0 software The mean±SEM is given (n=4).

[0111] Conclusion: Binding of ACZ885 to IL-1 beta is competitive withboth IL-1 receptor type I and type II.

[0112] Reactivity Profile to Human IL-1alpha, human IL-1RA, and IL-1betafrom Other Species

[0113] The reactivity profile of ACZ885 to human IL-1alpha, IL-1RA, andcynomolgous, rabbit, murine and rat IL-1beta is determined by Biacoreanalysis. ACZ885 is immobilized, and the cytokines examined are appliedat a concentration of 8 nM (6 independent runs.) TABLE 3 Crossreactivityof NVP-ACZ885 with IL-1β, IL-1α, and IL-1Ra % Binding (mean +/−SEM) RecHuman IL-1β (n = 6) 100 Rec Cynomolgus IL-β (n = 11) 7.8 +/− 1.0 RecRabbit IL-1β (n = 6) −0.5 +/− 0.2   Rec Mouse IL-1β (n = 6) −2.6 +/−0.6   Rec Rat IL-1β (n = 6) −6.2 +/− 1.0   Rec Human IL-1 α (n = 6) 8.4+/− 2.4 Rec Human IL-1Ra (n = 6) −3.7 +/− 1.7  

[0114] Resonance units were read at 1000 s after injection start; aninjection of running buffer was subtracted from all sensorgrams, and thebaseline after immobilization of anti-Fcγ set to zero. Binding isexpressed as percentage of accumulated resonance units for human IL-1β.

[0115] Conclusion: ACZ885 does not significantly crossreact with humanIL-1alpha, human IL-1RA, or cynomolgous, rabbit, murine or rat IL-1beta.

Example 3

[0116] Neutralization of the Release of IL-6 from Human DermalFibroblasts by ACZ885

[0117] The following methodology was used to assess the biologicalactivity of ACZ885 in neutralizing the action of human IL-1β:

[0118] 1. Preparation of Conditioned Medium Containing IL-1β

[0119] The preparation of conditioned medium from human peripheral bloodmononuclear cells was done as follows: mononuclear cells were preparedfrom the peripheral blood of monkeys using ficoll-hypaque densityseparation according to the method of Hansel [Hansel, T. T. et. al.(1991). An improved immunomagnetic procedure for the isolation of highlypurified human blood eosinophils. J. Imm. Methods 0.145: 105-110]; theywere used at a concentration of 10⁵ cells/well in RPMI/10% FCS. IFNβ(100 U/ml) and LPS (5 μg/ml) were added and cells were subsequentlyincubated for 6 hrs. Incubation was terminated by centrifugation at 1200RPM for 10 min. IL-1β in the supernatant was quantified using an ELISA

[0120] 2. Neutralization Assay

[0121] Human dermal foreskin fibroblasts were obtained from Clonetics(CC-2509) and grown in FBM (Clonetics, CC-3131) including bFGF (1 ng/ml,CC-4065), insulin (5 βg/ml, CC-4021), and 2% FCS (CC-4101).

[0122] For induction of IL-6, cells were seeded at a density of 10⁴cells per well in a 48 well tissue cluster. The following day, cellswere starved for 6-7 h in FBM containing 2% FCS before addition ofcytokine. For stimulation, the culture medium was replaced by FBM+2% FCScontaining the appropriate amount of conditioned medium for about 50pg/ml IL-1β. Alternatively, recombinant human IL-1β at a finalconcentration of 50 pg/ml was used.

[0123] Neutralizing anti-IL-1β antibody was titrated into the dilutedconditioned medium prior to addition to the cells. Recombinant IL-1Ra(R&D Systems # 280-RA-010) was used as a positive control.

[0124] Cell supernatant was taken 16-17 h after stimulation and theamount of released IL-6 determined in a sandwich ELISA.

[0125] 3. IL-6 ELISA

[0126] ELISA microtiter plates were coated with a murine anti-human IL-6MAb (314-14 (Novartis Pharma; batch EN23,961, 5.5 mg/ml); 100 III at 3μg/ml) in PBS 0.02% NaN₃ and incubated overnight at +4° C., Thefollowing day, microtiter plates were washed 4 times with PBS/0.05%Tween/0.02% NaN₃ and blocked with 300 μl of PBS/3% bovine serum albumin(BSA)/0.02% NaN₃ for 3 h. Plates were washed again (4 times) and 100 μlof supernatant (final dilutions of 1:20) or of the recombinant humanIL-6 standard ((Novartis Pharma #91902), titration curve ranging from 1to 0.0156 ng/ml in 2 fold dilution steps) was added in duplicate. Afteran overnight incubation at RT the plates were washed (4 times) and adifferent murine anti-human IL-6 MAb (110-14, Novartis Pharma; 6.3mg/ml); 100 μl at 1 μg/ml; 3 h at room temperature) was added. Afteradditional 4 washes, a biotin-labelled goat anti-mouse IgG2b antiserum(Southern Biotechnology; #1090-08) was added at the final dilution of{fraction (1/10000)} (100 μl/well; 3 h at room temperature). Afterincubation plates were washed 4 times and streptavidin coupled toalkaline phosphatase (Jackson Immunoresearch, #016-050-084) was added ata final dilution of {fraction (1/3000)} (100 μl/well; 30 min at roomtemperature). After washing (4 times) the substrate(p-nitrophenylphosphate in diethanolamine buffer; 100 μl) was added for30 min. Reaction was blocked by the addition of 50 μl/well of 1. 5 MNaOH. Plates were read in a microtiter reader (Bio-Rad) using filters of405 and 490 nm.

[0127] IL-6 levels in culture supernatants were calculated in referenceto the standard curve using the cubic curve fit. Statistical evaluationand determination of IC₅₀ was performed based on sigmoidal curvefitting. TABLE Inhibition of IL-1β-induced IL-6 secretion NVP-ACZ885NVP-ACZ885 IL-1ra Batch1 Batch2 IC₅₀ [pM] ± SEM IC₅₀ [pM] ± IC₅₀ [pM] ±SEM SEM IL-6 secretion 54 ± 6.1 44.6 ± 3.6 30 ± 3.1 cond. medium (9.1 ±1.0 ng/ml) (7.4 ± 0.6 (0.51 ± 0.05 ng/ml) ng/ml) (n = 6) (n = 6) (n = 5)IL-6 secretion 42 ± 3.4 63 ± 2.8 nd rec. human IL-1β (7.1 ± 0.56 ng/ml)(10.5 ± 0.5) (n = 4) (n = 6)

[0128] IC₅₀ values for inhibition of IL-1β-induced secretion of IL-6from human dermal fibroblasts. Fibroblasts were stimulated withrecombinant human IL-1β or conditioned medium containing between 50 and100 pg/ml of IL-15.

EXAMPLE 4

[0129] Definition of the Epitope for ACZ885

[0130] ACZ885 binds to human IL-1β with high affinity, but fails torecognize the highly homologous IL-1β derived from rhesus monkeys. Oneof the most prominent differences in the amino acid sequences betweenrhesus and human IL-1β is in position 64 of the mature IL-1β. HumanEL-10 has a glutamic acid, and rhesus an alanine in this position. Amutant human IL-1β with the respective replacement Glu64Ala has lost itsability to bind to ACZ885 with measurable affinity. We conclude thatGlu64 in human IL-1β is essential for recognition by the antibodyACZ885. Glu64 is located on a loop of IL-1β which is not part of thebinding surface to the IL-1β type I receptor, or in close proximity toit. Thereby, antibodies directed against a binding epitope incorporatingGlu64 have the potential to neutralize the biological activity of humanIL-1β.

1 4 1 137 PRT Homo sapiens 1 Met Glu Phe Gly Leu Ser Trp Val Phe Leu ValAla Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu SerGly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys AlaAla Ser Gly Phe Thr Phe 35 40 45 Ser Val Tyr Gly Met Asn Trp Val Arg GlnAla Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Ile Ile Trp Tyr Asp GlyAsp Asn Gln Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr IleSer Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Gly LeuArg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Leu ArgThr Gly Pro Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr ValSer Ser 130 135 2 126 PRT Homo sapiens 2 Met Leu Pro Ser Gln Leu Ile GlyPhe Leu Leu Leu Trp Val Pro Ala 1 5 10 15 Ser Arg Gly Glu Ile Val LeuThr Gln Ser Pro Asp Phe Gln Ser Val 20 25 30 Thr Pro Lys Glu Lys Val ThrIle Thr Cys Arg Ala Ser Gln Ser Ile 35 40 45 Gly Ser Ser Leu His Trp TyrGln Gln Lys Pro Asp Gln Ser Pro Lys 50 55 60 Leu Leu Ile Lys Tyr Ala SerGln Ser Phe Ser Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly SerGly Thr Asp Phe Thr Leu Thr Ile Asn Ser 85 90 95 Leu Glu Ala Glu Asp AlaAla Ala Tyr Tyr Cys His Gln Ser Ser Ser 100 105 110 Leu Pro Phe Thr PheGly Pro Gly Thr Lys Val Asp Ile Lys 115 120 125 3 410 DNA Homo sapiens 3atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120tgtgcagcgt ctggattcac cttcagtgtt tatggcatga actgggtccg ccaggctcca 180ggcaaggggc tggagtgggt ggcaattatt tggtatgatg gagataatca atactatgca 240gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300caaatgaacg gcctgagagc cgaggacacg gctgtgtatt attgtgcgag agatcttagg 360actgggcctt ttgactactg gggccaggga accctggtca ccgtctcctc 410 4 378 DNAHomo sapiens 4 atgttgccat cacaactcat tgggtttctg ctgctctggg ttccagcctccaggggtgaa 60 attgtgctga ctcagtctcc agactttcag tctgtgactc caaaggagaaagtcaccatc 120 acctgccggg ccagtcagag cattggtagt agcttacact ggtaccagcagaaaccagat 180 cagtctccaa agctcctcat caagtatgct tcccagtcct tctcaggggtcccctcgagg 240 ttcagtggca gtggatctgg gacagatttc accctcacca tcaatagcctggaagctgaa 300 gatgctgcag cgtattactg tcatcagagt agtagtttac cattcactttcggccctggg 360 accaaagtgg atatcaaa 378

1. An IL-1β binding molecule which comprises an antigen binding sitecomprising at least one immunoglobulin heavy chain variable domain(V_(H)) which comprises in sequence hypervariable regions CDR1, CDR2 andCDR3, said CDR1 having the amino acid sequence Val-Tyr-Gly-Met-Asn, saidCDR2 having the amino acid sequenceIle-Ile-Trp-Tyr-AspGly-Asp-Asn-Gln-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly, andsaid CDR3 having the amino acid sequence Asp-Leu-Arg-Thr-Gly-Pro; anddirect equivalents thereof.
 2. An IL-1β binding molecule comprising atleast one immunoglobulin light chain variable domain (V_(L)) whichcomprises in sequence hypervariable regions CDR1′, CDR2′ and CDR 3′,said CDR1′ having the amino acid sequenceArg-Ala-Ser-Gln-Ser-Ile-Gly-Ser-Ser-Leu-His said CDR2′ having the aminoacid sequence Ala-Ser-Gln-Ser-Phe-Ser and said CDR3′ having the aminoacid sequence His-Gln-Ser-Ser-Ser-Leu-Pro and direct equivalentsthereof.
 3. An IL-1β binding molecule comprising both heavy (V_(H)) andlight chain (V_(L)) variable domains in which said IL-1β bindingmolecule comprises at least one antigen binding site comprising: a) animmunoglobulin heavy chain variable domain (V_(H)) which comprises insequence hypervariable regions CDR1, CDR2 and CDR3, said CDR1 having theamino acid sequence Val-Tyr-Gly-Met-Asn, said CDR2 having the amino acidsequenceIle-Ble-TrpTyr-AspGly-Asp-Asn-Gln-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly, andsaid CDR3 having the amino acid sequence Asp-Leu-Arg-Thr-Gly-Pro, and b)an immunoglobulin light chain variable domain (V_(L)) which comprises insequence hypervariable regions CDR1′, CDR2′ and CDR3′, said CDR1′ havingthe amino acid sequence Arg-Ala-Ser-Gln-Ser-Ble-Gly-Ser-Ser-Leu-His,said CDR2′ having the amino acid sequence Ala-Ser-Gln-Ser-Phe-Ser, andsaid CDR3′ having the amino acid sequenceGln-Gln-Arg-Ser-Asn-Trp-Met-Phe-Pro; and direct equivalents thereof. 4.An IL-1β binding molecule according to claim 1, 2 or 3 which is a humanantibody.
 5. An IL-1β binding molecule which comprises at least oneantigen binding site comprising either a first domain having an aminoacid sequence substantially identical to that shown in Seq. Id. No. 1starting with amino acid at position 1 and ending with amino acid atposition 118 or a first domain as described above and a second domainhaving an amino acid sequence substantially identical to that shown inSeq. Id. No. 2, starting with amino acid at position 1 and ending withamino acid at position
 107. 6. A first DNA construct encoding a heavychain or fragment thereof which comprises a) a first part which encodesa variable domain comprising alternatively framework and hypervariableregions, said hypervariable regions being in sequence CDR1, CDR2 andCDR3 the amino acid sequences of which are shown in Seq. Id. No. 1; thisfirst part starting with a codon encoding the first amino acid of thevariable domain and ending with a codon encoding the last amino acid ofthe variable domain, and b) a second part encoding a heavy chainconstant part or fragment thereof which starts with a codon encoding thefirst amino acid of the constant part of the heavy chain and ends with acodon encoding the last amino acid of the constant part or fragmentthereof, followed by a stop codon.
 7. A second DNA construct encoding alight chain or fragment thereof which comprises a) a first part whichencodes a variable domain comprising alternatively framework andhypervariable regions; said hypervariable regions being CDR1′ and CDR3′,the amino acid sequences of which are shown in Seq. Id. No. 2; thisfirst part starting with a codon encoding the first amino acid of thevariable domain and ending with a codon encoding the last amino acid ofthe variable domain, and b) a second part encoding a light chainconstant part or fragment thereof which starts with a codon encoding thefirst amino acid of the constant part of the light chain and ends with acodon encoding the last amino acid of the constant part or fragmentthereof followed by a stop codon.
 8. An expression vector able toreplicate in a prokaryotic or eukaryotic cell line which comprises atleast one DNA constructs according to claim 6 or claim
 7. 9. A processfor the product of an IL-1β binding molecule which comprises (i)culturing an organism which is transformed with an expression vectoraccording to claim 8 and (H) recovering the IL-1β binding molecule fromthe culture.
 10. An antibody to IL-1β which has antigen bindingspecificity for an antigenic epitope of mature human IL-1β whichincludes the loop comprising residue Glu 64 and which is capable ofinhibiting the binding of IL-1β to its receptor.
 11. i) Use of anantibody to IL-ID according to claim 10, for the treatment of an IL-1mediated disease or disorder; ii) a method for the treatment of an IL-1mediated disease or disorders in a patient which comprises administeringto the patient an effective amount of an antibody according to claim 10;iii) a pharmaceutical composition comprising an antibody to IL-1βaccording to claim 10, in combination with a pharmaceutically acceptableexcipient, diluent or carrier; and iv) use of an antibody to IL-1βaccording to claim 10, for the preparation of a medicament for thetreatment of an IL-1 mediated disease or disorder.